Contrasting effects of heat treatment and incubation temperature on germination and outgrowth of individual spores of nonproteolytic Clostridium botulinum bacteria

Abstract

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic Clostridium botulinum Eklund 17B. The times to germination (t(germ)), one mature cell (t(C1)), and two mature cells (t(C2)) were measured for individual unheated spores incubated at 8, 10, 15, or 22 degrees C and used to calculate the t(germ), the outgrowth time (t(C1) - t(germ)), and the first doubling time (t(C2) - t(C1)). Measurements were also made at 22 degrees C of spores that had previously been heated at 80 degrees C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80 degrees C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (t(C1)) and outgrowth (t(C1) - t(germ)) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.

FIG. 1.

Frequency distributions of lag time (t_C1) for single spores of nonproteolytic C. botulinum Eklund 17B. Spores were unheated and incubated in PYGS medium at 22, 15, 10, and 8°C or were heated at 80°C for 20 s (∼10-fold reduction) and incubated in PYGS medium at 22°C (HT-22°C).

FIG. 2.

Frequency distributions of germination times (t_germ) for single spores of nonproteolytic C. botulinum Eklund 17B. Spores were unheated and incubated in PYGS medium at 22, 15, 10, and 8°C or were heated at 80°C for 20 s (∼10-fold reduction) and incubated in PYGS medium at 22°C (HT-22°C).

FIG. 3.

Frequency distributions of times for outgrowth (t_C1 − t_germ) for single spores of nonproteolytic C. botulinum Eklund 17B. Spores were unheated and incubated in PYGS medium at 22, 15, 10, and 8°C or were heated at 80°C for 20 s (∼10-fold reduction) and incubated in PYGS medium at 22°C (HT-22°C).

FIG. 4.

Frequency distributions of doubling times (t_C2 − t_C1) for single spores of nonproteolytic C. botulinum Eklund 17B. Spores were unheated and incubated in PYGS medium at 22, 15, 10, and 8°C or were heated at 80°C for 20 s (∼10-fold reduction) and incubated in PYGS medium at 22°C (HT-22°C).

FIG. 5.

Median times for germination (t_germ), outgrowth (t_C1 − t_germ), and doubling (t_C2 − t_C1) obtained from replicate experiments at different temperatures and the best-fit regression line.